High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%-50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.